1. Volume 146, Issue 1, Pages 80-91 (July 2011)
Eukaryotic Origin-Dependent DNA Replication In Vitro Reveals Sequential Action of DDK and S-CDK Kinases 
Ryan C. Heller, Sukhyun Kang, Wendy M. Lam, Shuyan Chen, Clara S. Chan, Stephen P. Bell 
Cell 
Volume 146, Issue 1, Pages (July 2011)
DOI: /j.cell
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2. Cell  , 80-91DOI: ( /j.cell )
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3. Figure 1 An Assay for Replisome Assembly In Vitro
(A) Schematic of replisome assembly assay. ARS1 origin DNA was treated with three sequential incubations: step 1, Mcm2–7 loading in G1 extract supplemented with Cdc6; step 2, DDK phosphorylation of Mcm2–7; step 3, replisome assembly in S phase extract.
(B) Protein, substrate, and extract requirements for the replisome assembly assay. Replisome assembly assays were performed with or without DDK-inactivated yRH182 S phase extract (yRH182-S, lanes 1–4) or with α-factor- (G1), hydroxyurea (HU)-, or nocodazol (Noc)-arrested extracts made from yRH182 expressing active DDK and overexpressing Cdc45, Dpb11, Sld2, and Sld3 (lanes 5–10). Unless indicated, wild-type (WT) ARS1 DNA and Cdc6 were used in all reactions. ARS1-A-B2- is an ARS1 mutant lacking ORC-binding sites. Additional DDK (125 ng) was added to the 2nd extract in lanes 8–10. Changes in ORC DNA association were likely due to release of ORC after pre-RC formation (lanes 5 and 8; Tsakraklides and Bell, 2010) and ORC rebinding after Cdc45, GINS, and Mcm10 recruitment.
See also Table S2 and Table S3.
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4. Figure 2 Interdependent Recruitment of Replisome Proteins
Depletion of replication proteins reveals interdependent origin DNA association. S phase extracts were depleted for the indicated protein prior to replisome assembly assays. Associated proteins were analyzed by immunoblot. Depleted protein and extracts were as follows: Sld3, yRH208-S; Cdc45, yRH182-S; Sld2, yRH207-S; Dpb11, yRH209-S; GINS, yRH223-S; Mcm10, yRH183-S. For each panel, Cdc6 was omitted from reaction 1, S phase extracts were depleted for the indicated protein in reactions 3–4, and the corresponding purified protein (see Figure S1) was added in reaction 4. Note: purified Cdc45-FLAG and MBP-Mcm10 lack HA and myc tags, respectively. GINS was detected with a polyclonal antibody in lanes 25–28. See also Figure S1 and Figure S2 and Table S2 and Table S3.
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5. Figure 3
Long DNA Templates Support Polymerase Loading and Replication Initiation
(A) Circular templates show increased DNA Pol α association. Replisome assembly assays were performed with 1 kb linear ARS1 DNA or pARS1/WT plasmid.
Throughout this figure, lines and ovals below images indicate biotinylated linear and circular templates, respectively.
(B) Analysis of replication products. Replication assays were performed using yRH182-S extract on pARS1/WT template. Left, native gel of DNA products, ethidium bromide stain. The location of relaxed plasmid is indicated. Center, autoradiogram of the native gel. Right, autoradiogram of replication products analyzed by alkaline gel electrophoresis. The presence of Cdc6 during Mcm2–7 loading is indicated.
(C) Protein, template, and nucleotide requirements of the replication assay. Replication assays were performed with yRH182-S extract. Reactions lacking Cdc6 during helicase loading are indicated. Immunoblot (upper panels) and alkaline gel analysis (lower panels) of proteins and replication products are shown. Templates used: lanes 1–4, circular pARS1/WT (5.6 kb); lanes 5–7, circular pUC19-ARS1 (3.7 kb); lanes 8 and 9, circular pARS1/Nco-Nco (7.6 kb); lanes 10 and 11, linear pARS1/Nco-Nco (7.6 kb). Lanes 7, 9, and 11 use A-B2- derivatives of the indicated DNA. ATPγS reactions replaced ATP and the ATP-regenerating system with 1 mM ATPγS in step 3 of the assay. + aphid, 100 μg/ml aphidicolin in step 3.
(D) Timecourse of Mcm10 recruitment and replication product accumulation. Replication assays using yRH182-S extract and pARS1/WT were analyzed by immunoblot of origin-associated proteins (upper panels) and nucleotide incorporation (lower panel).
(E and F) Relative contributions of overexpressed Cdc45 (C45), Dpb11 (D11), Sld2 (S2), and Sld3 (S3). Replication assays using pARS1/WT plasmid template and yRH182-S (lane 1) or yRH191-S (no overexpressed proteins, lanes 2–9) extracts were supplemented with the indicated purified replication proteins. Relative level of replication products (lower panel) was quantified and plotted in (F).
(G) Heavy-light analysis of replication products. Replication reactions were performed in the presence of 500 μM BrdUTP in place of dTTP. Replication products were fractionated by CsCl gradient and detected by scintillation counter (black line). Heavy-heavy and light-light controls are shown (gray line). The CsCl density (g/ml) of the highest point in each peak is indicated.
See also Figure S1 and Table S2 and Table S3.
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6. Figure 4
DDK and S-CDK Are Required for Distinct Stages of Origin Activation
(A and B) S-CDK and DDK are required for replisome assembly. Replisome assembly assays using extracts yRH166-S (A) or yRH229-S (B) were analyzed as described in Figure 1. DDK kinase activity was eliminated by omitting DDK from reaction step 2. S-CDK activity was blocked by the addition of GST-Sic1 to reaction step 3. DDK with CDK, DDK was omitted from reaction step 2 and added to reaction step 3 in which S-CDK is also active. CDK→DDK, reaction step 2 was eliminated. After step 3, purified GST-Sic1 and DDK were added sequentially and incubation was continued for 20 min.
(C) S-CDK and DDK are required for DNA replication. Replication assays used yRH182-S and pARS1/WT plasmid template. Mcm4-Pi immunoblot was probed with the Mcm4-phospho-S82-D83 phosphospecific antibody that recognizes a DDK target site in Mcm4 (Randell et al., 2010).
(D) Sld3 binding to ARS305 in G1 requires DDK. Either wild-type CDC7 or congenic cdc7-4 strains including myc-tagged Sld3 were arrested in nocodazole and released into 25°C or 32°C media containing α-factor (Figure S3) and analyzed by ChIP using anti-Mcm2–7 or anti-myc antibodies. Samples were analyzed by PCR using primers recognizing ARS305 and two non-origin sequences (ARS305+17kb and ARS306+6kb) (Table S4).
(E) Cdc45 binding to early origins in G1 requires DDK. Either wild-type CDC7 or congenic cdc7-4 strains including myc-tagged Cdc45 were arrested in media containing α-factor at 25°C (Figure S3) and analyzed by ChIP-Chip using anti-myc antibodies. The average log2 ratios of immunoprecipitate (IP) to input signal from two experiments are plotted for chromosome III (wild-type, orange; cdc7-4, blue). Three early origins (ARS305, ARS306, and ARS307) and one late origin (ARS316) are indicated.
See also Figure S2 and Figure S3 and Table S1, Table S2, and Table S3.
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7. Figure 5
Mcm10 Is Required for the Recruitment of Pol α and Pol δ to Origin DNA
(A) Effect of Mcm10 depletion on DNA polymerase origin association. Replication assays were performed with pARS1/WT plasmid template and extract yRH183-S (lanes1–3), yRH185-S (lanes 4–6), or yRH187-S (lanes 7–9). As indicated, extracts were depleted of Mcm10 and supplemented with MBP-Mcm10.
(B) Relative levels of DNA polymerase association. Two (Pol α and Pol ɛ) or three (Pol δ) iterations of the experiment in (A) were quantified and plotted. Polymerase recruitment in the undepleted extract was set to 1. Error bars = standard deviation from the mean.
See also Figure S1 and Table S2 and Table S3.
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8. Figure 6
ATP Hydrolysis Is Required for RPA Loading and for the Loading of a Subset of Polymerases
(A) ATP hydrolysis requirement for RPA loading. Replisome assembly assays were performed using pARS1/WT plasmid templates and extract yRH184-S. Where indicated, ATPγS was added after DDK phosphorylation of Mcm2–7 in place of ATP and the ATP-regenerating system in reaction step 3.
(B) Cdc45 was required for RPA loading and DNA replication. Replication assays were performed using pARS1/WT templates and extract yRH188-S. As indicated, extract was depleted for Cdc45 and supplemented with purified Cdc45-3HA/3Flag.
(C) ATP hydrolysis requirement for DNA polymerase recruitment. Replisome assembly reactions were performed using pARS1/WT plasmid templates and extracts yRH188-S (lanes 1–4), yRH184-S (lanes 5–7), yRH186-S (lanes 8–10), or yRH182-S (lanes 11–13). ATP: Reactions were performed under standard conditions. +Sic1: GST-Sic1 added to reaction step 3. ATPγS+AS-CDK: During reaction step 3, ATP and the ATP-regenerating system were replaced with 1 mM ATPγS, analog-specific CDK (CDK-AS), and 0.5 mM 6-benzyl-ATP.
See also Figure S1 and Figure S4 and Table S2 and Table S3.
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9. Figure 7 A Model for the Events Leading to Replication Initiation
(I) Helicase loading. The Mcm2–7 helicase is loaded to origin DNA in an inactive form during late M/G1 phase of the cell cycle to form the pre-RC. (II) DDK-dependent complex formation. In late G1 or S phase, DDK targets Mcm2–7 for phosphorylation, allowing recruitment of Sld3 and Cdc45. (III) Helicase activation. S-CDK activation and phosphorylation of Sld2 and Sld3 trigger the recruitment of Sld2, Dpb11, GINS, and Pol ɛ, and the subsequent recruitment of Mcm10. The formation of the Cdc45-Mcm2–7-GINS complex activates the helicase, triggering melting of origin DNA. (IV) Complete replisome assembly. Pol α and Pol δ are loaded on the unwound DNA in an Mcm10-dependent process to complete replisome assembly.
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10. Figure S1
Purified Proteins Used in This Study, Related to Figure 1, Figure 2, Figure 3, and Figure 5
(A–E) Purified proteins were analyzed by SDS-PAGE.
(A) Coomassie blue stain of GINS complex purified from yeast strain yRH156, which overexpresses Sld5, Psf1, Psf2-Flag, and Psf3.
(B) Coomassie blue stain of proteins purified from the following yeast strains: Sld2-Flag, yRH152 (lane 1); Sld3-Flag, yRH153 (lane 2); Dpb11-Flag, yRH154 (lane 3).
(C) Coomassie blue stain of MBP-Mcm10 purified from E. coli Rosetta 2(DE3)(pLysS)(pRH121).
(D) Coomassie blue stain of Cdc45-3HA/3Flag purified from yeast strain ySK-Cdc45.
(E) Sypro Orange stain of DDK complex purified from yeast strain yLF52, which overexpresses Cdc7-proA and Dbf4-CBP.
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11. Figure S2
Cdc45 Origin Association Is Stabilized by S-CDK-Dependent Events, Related to Figure 2 and Figures 4A–4C
Cdc45 is more sensitive to salt extraction in the absence of S-CDK activity. Replisome assembly assays were performed with 1 kb linear ARS1 DNA template and yRH182 S phase extract under conditions where S-CDK activity is blocked by GST-Sic1 (+ Sic1) or unaltered (- Sic1) and then analyzed by immunoblot. Before UV-release of bead-bound proteins, the beads were washed an additional time with H buffer containing the indicated concentration of NaCl. Unless indicated, Cdc6 was added to the G1 extract used in step one of all reactions.
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12. Figure S3
Origins Bound by Cdc45 in G1 in a Cdc7-Dependent Manner Initiate Early in S phase, Related to Figures 4E and 4F
(A and B) Replication timing of origins that bound Cdc45 in a Cdc7-dependent manner during G1. We identified 49 Cdc7-dependent sites of Cdc45-binding in G1-arrested cells (Table S1), all of which correspond to OriDB “confirmed” or “likely” origins (Nieduszynski et al., 2007). For heavy:light (A, Raghuraman et al., 2001) or copy-number (B, Yabuki et al., 2002) replication timing studies, we plotted a histogram of the Trep numbers for these sites (in orange), and superimposed this over the histogram of the Trep numbers at all 562 OriDB “confirmed” or “likely” origins (in blue). Histogram bin sizes are 2 min each.
(C) FACS analysis of cells prior to Cdc45 and Sld3 ChIP. Top: FACS profiles of ySC331 (sld3-13myc cdc7-4) and ySC332 (sld3-13myc CDC7) prior to and after the nocodazol and α-factor arrest (at 25°C and 32°C). Bottom: FACS profiles of yWL17 (cdc45-13myc cdc7-4) and yWL18 (cdc45-13myc CDC7) before and after α-factor arrest.
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13. Figure S4
Cdc45 and Sld3 Do Not Require DNA Unwinding for Recruitment, Related to Figure 6
Sld3 and Cdc45 are recruited to origin DNA when ATPγS is present in step 3 of the replisome assembly assay. Immunoblot analysis of replisome assembly reaction utilizing 1 kb linear ARS1 DNA template and extract yRH229-S. Cdc6 was present or omitted as indicated. In the ATPγS reaction (lane 3), step three incubation contained 1 mM ATPγS instead of ATP and the ATP-regenerating system.
Cell  , 80-91DOI: ( /j.cell )
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